Fanconi Anemia (FA) is an autosomal recessive cancer susceptibility disorder characterized by congenital abnormalities, bone marrow failure, and cellular hypersensitivity to DNA crosslinking agents. Nine FA genes have been cloned, and the nine encoded proteins (A, B, C, D1, D2, E, F, G, L) cooperate in a common cellular pathway, leading to the monoubiquitination of FANCD2 and its interaction in chromatin with FANCD1/BRCA2. Interestingly, a novel deubiquitinating enzyme, called USP1, deubiquitinates FANCD2 and shuts the pathway "off1 (Nijman et al, Molecular Cell 17: 331,2005). Little is known about the regulation of USP1, the other substrates of USP1, or the global cellular function of USP1 in DNA repair. Very recently, we have made two important observations about USP1. First, USP1 is rapidly degraded following DNA damage, resulting in the accumulation of FANCD2-Ub. This result suggests that FANCD2-Ub accumulation results from decreased deubiquitination as well as increased ubiquitination by the FA complex. Second, in addition to FANCD2-Ub, USP1 also deubiquitinates the processivity factor, PCNA, and thus regulates translesion DNA synthesis (TLS). This observation suggests a functional connection between the FA pathway and DNA replication by variant DNA polymerases in TLS. Based on these new observations, we propose the following three aims for the next five years. In Specific Aim #1, we will examine the molecular mechanism of DNA damage-inducible USP1 degradation. In Specific Aim #2, we will examine the cellular outcome of USP1 dysregulation (namely, the affect on FA pathway mediated and TLS-mediated DNA repair activity and mutation frequency). For this aim, we will use a newly-generated Usp1-deficient mouse model. In Specific Aim #3, we will examine the monoubiquitination and deubiquitination of FANCD2 mutant polypeptides in vitro and in vivo. We will also determine the interaction of these FANCD2 mutant proteins with the FA complex and with USP1. Overall, we believe these studies will elucidate the cellular regulatory mechanisms which control the monoubiquitination state of key biological modulators, such as FANCD2.